The present invention pertains in general to hematopoietic growth factors and to polynucleotides encoding such factors. The present application pertains in particular to mammalian pluripotent colony stimulating factors, specifically human pluripotent granulocyte colony-stimulating factor (hpG-CSF), to fragments and polypeptide analogs thereof and to polynucleotides encoding the same.
The human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets). The hematopoietic system of the average human male has been estimated to produce on the order of 4.5.times.10.sup.11 granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight. Dexter et al., BioEssays, 2, 154-158 (1985).
It is believed that small amounts of certain hematopoietic growth factors account for the differentiation of a small number of progenitor "stem cells" into the variety of blood cell lines, for the tremendous proliferation of those lines, and for the ultimate differentiation of mature blood cells from those lines. Because the hematopoietic growth factors are present in extremely small amounts, the detection and identification of these factors has relied upon an array of assays which as yet only distinguish among the different factors on the basis of stimulative effects on cultured cells under artificial conditions. As a result, a large number of names have been coined to denote a much smaller number of factors. As an example of the resultant confusion the terms, IL-3, BPA, multi-CSF, HCGF, MCGF and PSF are all acronyms which are now believed to apply to a single murine hematopoietic growth factor. Metcalf, Science, 229, 16-22 (1985). See also, Burgess, et al. J. Biol. Chem., 252, 1998-2003 (1977), Das, et al. Blood, 58, 630-641 (1981), Ihle, et al., J. Immunol., 129, 2431 (1982), Nicola, et al., J. Biol. Chem., 258, 9017 (1983), Metcalf, et al., Int. J. Cancer, 30, 773 (1982), and Burgess, et al. Int. J. Cancer, 26, 647 (1980), relating to various murine growth regulatory glycoproteins.
The application of recombinant genetic techniques has brought some order out of this chaos. For example, the amino acid and DNA sequences for human erythropoietin, which stimulates the production of erythrocytes, have been obtained. (See, Lin, PCT Published Application No. 85/02610, published Jun. 20, 1985.) Recombinant methods have also been applied to the isolation of cDNA for a human granulocyte-macrophage colony-stimulating factor. See, Lee, et al., Proc. Natl. Acad. Sci. (U.S.A.), 82, 4360-4364 (1985) and Wong, et al., Science, 228, 810-814 (1985). See also Yokota, et al. Proc. Natl. Acad. Sci. (U.S.A.), 81, 1070 (1984), Fung, et al., Nature, 307, 233 (1984), and Gough, et al., Nature, 389, 763 (1984) relating to cloning of murine genes, as well as Kawasaki, et al., Science, 230, 291 (1985) relating to human M-CSF.
A human hematopoietic growth factor, called human pluripotent colony-stimulating factor (hpCSF) or pluripoietin, has been shown to be present in the culture medium of a human bladder carcinoma cell line denominated 5637 and deposited under restrictive conditions with the American Type Culture Collection, Rockville, Md. as A.T.C.C. Deposit No. HTB-9. The hpCSF purified from this cell line has been reported to stimulate proliferation and differentiation of pluripotent progenitor cells leading to the production of all major blood cell types in assays using human bone marrow progenitor cells. Welte et al., Proc. Natl. Acad. Sci. (U.S.A.), 82, 1526-1530 (1985). Purification of hpCSF employed: (NH.sub.4).sub.2 SO.sub.4 precipitation; anion exchange chromatography (DEAE cellulose, DE52); gel filtration (AcA54 column); and C18 reverse phase high performance liquid chromatography. A protein identified as hpCSF, which is eluted in the second of two peaks of activity in C18 reverse phase HPLC fractions, was reported to have a molecular weight (MW) of 18,000 as determined by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) employing silver staining. HpCSF was earlier reported to have an isoelectric point of 5.5 [Welte, et al., J. Cell. Biochem., Supp 9A, 116 (1985)] and a high differentiation activity for the mouse myelomonocytic leukemic cell line WEHI-3B D.sup.+ [Welte, et al., UCLA Symposia on Molecular and Cellular Biology, Gale, et al., eds., New Series, 28 (1985)]. Preliminary studies indicate that the factor identified as hpCSF has predominately granulocyte colony-stimulating activity during the first seven days in a human CFU-GM assay.
Another factor, designated human CSF-.beta., has also been isolated from human bladder carcinoma cell line 5637 and has been described as a competitor of murine .sup.125 I-labelled granulocyte colony-stimulating factor (G-CSF) for binding to WEHI-3B D.sup.+ cells in a dose-response relationship identical to that of unlabelled murine G-CSF [Nicola, et al., Nature, 314, 625-628 (1985)]. This dose-response relationship had previously been reported to be unique to unlabelled murine G-CSF and not possessed by such factors as M-CSF, GM-CSF, or multi-CSF [Nicola, et al., Proc. Natl. Acad. Sci. (U.S.A.), 81, 3765-3769 (1984)]. CSF-.beta. and G-CSF are also unique among CSF's in that they share a high degree of ability to induce differentiation of WEHI-3B D.sup.+ cells. Nicola, et al., Immunology Today, 5, 76-80 (1984). At high concentrations, G-CSF stimulates mixed granulocyte/macrophage colony-forming cells [Nicola, et al., (1984) supra], which is consistent with preliminary results indicating the appearance of granulocytic, monocytic, mixed granulocytic/monocytic and eosinophilic colonies (CFU-GEMM) after 14 days incubation of human bone marrow cultures with hpCSF. CSF-.beta. has also been described as stimulating formation of neutrophilic granulocytic colonies in assays which employed mouse bone marrow cells, a property which has been a criterion for identification of a factor as a G-CSF. On the basis of these similarities, human CSF-.beta. has been identified with G-CSF (granulocytic colony stimulating factor). Nicola et al., Nature, 314, 625-628 (1985).
Based upon their common properties, it appears that human CSF-.beta. of Nicola, et al., supra, and the hpCSF of Welte, et al., supra, are the same factor which could properly be referred to as a human pluripotent granulocyte colony-stimulating factor (hpG-CSF). Characterization and recombinant production of hpG-CSF would be particularly desirable in view of the reported ability of murine G-CSF to completely suppress an in vitro WEHI-3B D.sup.+ leukemic cell population at "quite normal concentrations", and the reported ability of crude, injected preparations of murine G-CSF to suppress established transplanted myeloid leukemias in mice. Metcalf, Science, 229, 16-22 (1985). See also, Sachs, Scientific American, 284(1), 40-47 (1986).
To the extent that hpG-CSF may prove to be therapeutically significant and hence need to be available in commercial scale quantities, isolation from cell cultures is unlikely to provide an adequate source of material. It is noteworthy, for example, that restrictions appear to exist against commercial use of Human Tumor Bank cells such as the human bladder carcinoma cell line 5637 (A.T.C.C. HTB9) which have been reported as sources of natural hpCSF isolates in Welte, et al. (1985, supra).